Mutant glycine oxidase derived from thermophilic bacterium belonging to family bacillus, and method for producing same

ABSTRACT

A mutant glycine oxidase is obtained by substituting at least one wild-type amino acid sequence derived from thermophilic bacteria belonging to the family Bacillus with another amino acid, and has the following enzyme properties. Molecular weight: 40,000±2,000 daltons by SDS-PAGE. Optimum temperature: 45° C. under the condition of pH 8.5 in presence of pyrophosphate. Optimum pH: pH 8.0 under the condition of 37° C. in presence of pyrophosphate. Thermal stability: Stable up to 70° C. under the condition of pH 8.5 while retaining for 1 hour in presence of pyrophosphate. pH Stability: Stable in the range of pH 5.5 to 10.0 under the condition of 4° C. while retaining for 24 hours in presence of pyrophosphate. Specific activity: 1.2 units/mg or more. Kinetic constant Km: 0.2 mM or less.

TECHNICAL FIELD

The present invention relates to a mutant glycine oxidase derived fromthermophilic bacteria belonging to the family Bacillus and a method forproducing the same, and in particular, relates to a mutant glycineoxidase derived from thermophilic bacteria belonging to the familyBacillus and a method for producing the same, capable of achieving bothgood thermal stability and good enzyme activity.

BACKGROUND ART

Glycine is an amino acid having the simplest structure that has nostereoisomerism of D-form or L-form, and is one of the amino acids thatconstitute proteins, and it is also known as a raw material (startingmaterial) for biosynthesis of various biological substances. As one ofthe methods for measuring glycine, a mechanical measurement method usingmass spectrometry (MS), high performance liquid chromatography (HPLC),an amino acid analyzer or the like is known. However, in the mechanicalmeasurement method, in general, a measuring instrument is expensive andmaintenance cost is high, and operation requires skill.

Therefore, an enzyme measurement method using glycine oxidase that actson glycine and the like have been proposed as a cheaper and simplermeasurement method. One of typical glycine oxidases is known to bederived from Bacillus subtilis. For example, Non-PTL 1 has specificallyreported on a glycine oxidase derived from Bacillus subtilis.

In addition, examples of glycine oxidase currently on the market includeproduct number H244K manufactured by BioVision, Inc. This commerciallyavailable glycine oxidase is also derived from Bacillus subtilis, and isa mutant enzyme in which a mutation has been introduced into a wild-typeglycine oxidase. Non-PTL 2 has specifically reported on the mutantglycine oxidase and also proposes a biosensor using the same.

Further, PTL 1 discloses a modified glycine oxidase in which at leastone of amino acid residues is displaced to modify properties such asenzyme activity, thermal stability, and substrate specificity, and amethod for analyzing glycine using the same. The modified glycineoxidase specifically disclosed in the Examples of PTL 1 is also derivedfrom Bacillus subtilis, and is obtained by introducing a mutation of anamino acid residue into a wild-type glycine oxidase.

CITATION LIST Patent Literature

-   PTL 1: WO 2014/157705 A

Non-Patent Literature

-   Non-PTL 1: Yoshiaki Nishiya, Tadayuki Imanaka, “Purification and    characterization of a novel glycine oxidase from Bacillus subtilis”    FEBS (Federation of European Biochemical Societies) Letter Vol. 438    pp. 263-266 (1998)-   Non-PTL 2: Elena Rosini, Luciano Piubelli, Gianluca Molla, Luca    Frattini, Mattia Valentino, Antonio Varriale, Sabato D′Auria and    Loredano Pollegioni, “Novel biosensors based on oprimized glycine    oxidase” FEBS (Federation of European Biochemical Societies) Journal    Vol. 281 pp. 3460-3472 (2014)

SUMMARY OF INVENTION Technical Problem

The modified glycine oxidase disclosed in PTL 1 certainly allowsmodification of properties such as enzyme activity, thermal stability,and substrate specificity, with respect to the wild-type. For example,its specific activity and the like are about the same as those ofcommercially available glycine oxidase.

In addition, in recent years, in order to improve industrialapplicability, various enzymes are required to have thermal stability(or heat resistance) that can maintain good enzyme activity even underhigher temperature conditions. PTL 1 also attempts to improve thermalstability by modifying a wild-type glycine oxidase, but recently, thereis a tendency to require even better thermal stability.

The present invention has been made to solve such problems, and anobject of the present invention is to provide a mutant glycine oxidasederived from bacteria and a method for producing the same, capable ofachieving good thermal stability as well as good enzyme activity.

Solution to Problem

In order to solve the above-described problem, the mutant glycineoxidase according to the present invention is a mutant glycine oxidasewhich is a mutant enzyme in which at least one amino acid sequence in awild-type glycine oxidase derived from thermophilic bacteria belongingto the family Bacillus is substituted with another amino acid, and hasthe following configurations: the molecular weight is 40,000±2,000daltons in SDS-polyacrylamide gel electrophoresis, the optimumtemperature is 45° C. under a condition of pH 8.5 in presence ofpyrophosphate; the optimum pH is pH 8.0 under a condition of 37° C. inpresence of pyrophosphate; thermal stability is stable up to 70° C.under a condition of pH 8.5 while retaining for 1 hour in presence ofpyrophosphate; pH stability is stable in the range of pH 5.5 to 10.0under a condition of 4° C. while retaining for 24 hours in presence ofpyrophosphate; the specific activity is 1.2 units/mg or more; and thekinetic constant (Michaelis constant) K_(m) is 0.2 mM or less.

According to the above configurations, the mutant glycine oxidaseachieves the above enzyme properties by introducing a mutation into thewild-type glycine oxidase derived from thermophilic bacteria belongingto the family Bacillus. This mutant glycine oxidase can maintain akinetic constant K_(m) that is substantially in the same range as thatof the conventional mutant glycine oxidase, and also can achieve highthermal stability as compared not only with the conventional mutantglycine oxidase but also with the wild-type glycine oxidase, and furtherachieves high specific activity as compared with the conventional mutantglycine oxidase and the wild-type glycine oxidase. Thereby, in themutant glycine oxidase, it is possible to achieve good thermal stabilityas well as good enzyme activity.

In the mutant glycine oxidase of the above configuration, a glycine in apartial amino acid sequence that binds in the order of asparagine (N),glycine (G), cysteine (C), and tyrosine (Y) contained in the amino acidsequence of the wild-type glycine oxidase is substituted with anotheramino acid may be configured.

Also, in order to solve the above-described problem, the mutant glycineoxidase according to the present invention may be configured as a mutantenzyme of glycine oxidase derived from thermophilic bacteria belongingto the family Bacillus showing 95% or more homology to an amino acidsequence represented by SEQ ID NO: 1, having an amino acid sequence inwhich a 251st amino acid in the amino acid sequence represented by SEQID NO: 1 is substituted from glycine to another amino acid.

According to the above configuration, the mutant glycine oxidase is oneobtained by introducing a mutation into the wild-type glycine oxidasederived from thermophilic bacteria belonging to the family Bacillus atthe above position. This mutant glycine oxidase can maintain a kineticconstant K_(m) that is substantially in the same range as that of theconventional mutant glycine oxidase, and also can achieve high thermalstability as compared not only with the conventional mutant glycineoxidase but also with the wild-type glycine oxidase, and furtherachieves high specific activity as compared with the conventional mutantglycine oxidase and the wild-type glycine oxidase. Thereby, in themutant glycine oxidase, it is possible to achieve good thermal stabilityas well as good enzyme activity.

In addition, in the mutant glycine oxidase of the above configuration,it may be a configuration having an amino acid sequence in which the251st amino acid in the amino acid sequence represented by SEQ ID NO: 1is substituted from glycine to a basic amino acid or a hydrophobic aminoacid.

Moreover, in the mutant glycine oxidase of the above configuration, itmay be configured that the basic amino acid is glutamine, arginine, orhistidine, and the hydrophobic amino acid is isoleucine or threonine.

Further, in the mutant glycine oxidase of the above configuration, itmay be configured that the another amino acid is alanine, glutamic acid,histidine, isoleucine, asparagine, glutamine, arginine, or threonine.

Furthermore, in the mutant glycine oxidase of the above configuration,it may be configured that the thermophilic bacteria belonging to thefamily Bacillus is a bacteria belonging to the genus Bacillus, the genusAlicyclobacillus, the genus Brevibacillus, the genus Geobacillus, thegenus Sulfobacillus, the genus Paenibacillus, or the genus Salinicoccus.

Also, the present invention includes a DNA encoding the mutant glycineoxidase of the above configuration.

In addition, the present invention also includes a replicablerecombinant DNA containing the DNA of the above configuration and anautonomously replicable vector.

Moreover, the present invention also includes a cell obtained byintroducing the DNA of the above configuration or the recombinant DNA ofthe above configuration into a host cell.

Further, the present invention also includes a method for producing amutant glycine oxidase including culturing the cells of the aboveconfiguration, and collecting the mutant glycine oxidase from theresulting culture.

The above object, other objects, features, and advantages of the presentinvention will become apparent from the following detailed descriptionof preferred embodiments with reference to the accompanying drawings.

SEQUENCE LISTING

The nucleotide and amino acid sequences for the wild-type and mutantglycine oxidase described herein are listed in the sequence listingentitled “Mutated Glycine Oxidase derived from Bacillus family,” createdon Dec. 17, 2020, and having a file size of 11 KB, the entirety of whichis hereby incorporated by reference for all purposes.

Advantageous Effects of Invention

In the present invention, an effect that a mutant glycine oxidasederived from bacteria and a method for producing the same, capable ofachieving good thermal stability as well as good enzyme activity, can beprovided is exhibited by the above configuration.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a sequence diagram showing SEQ ID NO: 1, which is an aminoacid sequence of a wild-type glycine oxidase exemplified in anembodiment of the present invention, with a mutation corresponding toSEQ ID NO: 3 indicated in the dashed box.

FIG. 2 is a sequence diagram showing SEQ ID NO: 2, which is a nucleotidebase sequence of DNA encoding the wild-type glycine oxidase having theamino acid sequence of FIG. 1 , with a mutation corresponding to SEQ IDNO: 4 indicated in the dashed box.

FIG. 3A is a graph comparing specific activities of each of glycineoxidases in example and comparative examples, and FIG. 3B is a graphcomparing thermal stability of each of glycine oxidases in example andcomparative examples.

DESCRIPTION OF EMBODIMENTS

The mutant glycine oxidase derived from bacteria according to thepresent disclosure is a mutant enzyme in which at least one of the aminoacid sequences in the wild-type glycine oxidase derived fromthermophilic bacteria belonging to the family Bacillus is substitutedwith another amino acid, and it has characteristic enzyme propertieseven compared with the conventional glycine oxidase.

[Thermophilic Bacteria Belonging to Family Bacillus]

The thermophilic bacteria belonging to the family Bacillus from whichthe mutant glycine oxidase according to the present disclosure isderived is not particularly limited as long as they are bacteria thatare classified as Bacillaceae and have thermophilicity. The term“thermophilicity” as used herein means that the optimum growthtemperature of the bacteria is 45° C. or more or the growth limittemperature of the bacteria is 55° C. or more, or both are satisfied.

Specific examples of the bacteria belonging to the family Bacillusinclude bacteria belonging to the genus Bacillus, the genusAlicyclobacillus, the genus Anoxybacillus, the genus Brevibacillus, thegenus Geobacillus, the genus Halobacillus, the genus Oceanobacillus, thegenus Paenibacillus, the genus Sulfobacillus, the genus Virgibacillus,or the genus Salinicoccus, but are not particularly limited.

In the examples described later, Geobacillus kaustophilus HTA426 strainis used as the thermophilic bacteria belonging to the family Bacillus. Awild-type glycine oxidase derived from this Geobacillus kaustophilusHTA426 strain has a characteristic region having high homology with theamino acid sequences of glycine oxidases derived from each ofGeobacillus stearothermophilus, Geobacillus thermoleovorans, Geobacillusthermodenitrificans and Geobacillus subterraneus, which are otherthermophilic bacteria belonging to the family Bacillus.

As a commercially available glycine oxidase, a mutant enzyme derivedfrom Bacillus subtilis, which is a non-thermophilic Bacillus bacterium,is known as described above. For convenience of explanation, among theglycine oxidases derived from Bacillus subtilis, the wild type isreferred to as “B. subtilis wild-type glycine oxidase”, and the mutanttype is referred to as “B. subtilis mutant glycine oxidase”. Moreover,the glycine oxidase derived from the Geobacillus kaustophilus (G.kaustophilus) strain HTA426 strain is also referred to as “G.kaustophilus wild-type glycine oxidase” for convenience of explanation.

The three-dimensional structure of B. subtilis wild-type glycine oxidasehas been reported, for example, in Non-PTL 2, and the three-dimensionalstructure of G. kaustophilus wild-type glycine oxidase has beenreported, for example, in Reference Literature 1: Takako Shiono, TakaomiNomura, Yoshiaki Nishiya, Ryoichi Arai, “Crystal structure of glycineoxidase from Geobacillus kaustophilus” Photon Factory Activity Report2014 #32, PART B, Users' Report (Biological Science, No. 204), 2015.

In B. subtilis wild-type glycine oxidase, seven types of motifs areknown, and for example, in the examples of Non-PTL 2 and PTL 1,mutations are introduced into one of these motifs, an HCY motif. The HCYmotif is located in a pathway where a substrate such as glycine goes tothe active center of glycine oxidase.

While G. kaustophilus wild-type glycine oxidase has thermal stabilitysuperior to that of B. subtilis mutant glycine oxidase, as demonstratedexperimentally in the examples described later, the enzyme activity isgreatly inferior.

Therefore, the present inventors have considered that enzyme activitycan be improved while maintaining thermal stability when a mutation isalso introduced into the wild-type HCY motif or at a positioncorresponding to the HCY motif, also in the G. kaustophilus wild-typeglycine oxidase, similarly to the B. subtilis mutant glycine oxidase,and intensively studied. However, when comparing the amino acidsequences between the G. kaustophilus wild-type glycine oxidase and theB. subtilis wild-type glycine oxidase, it has been revealed that the G.kaustophilus wild-type glycine oxidase has a characteristic region thatis not be seen in the B. subtilis wild-type glycine oxidase.

As shown in FIG. 1 , this characteristic region is a partial amino acidsequence that binds in the order of a 250th asparagine (N), a 251stglycine (G), a 252nd cysteine (C), and a 253rd tyrosine (Y) in the aminoacid sequence of the G. kaustophilus wild-type glycine oxidase. Inaddition, it has been also revealed that this characteristic region hashigh homology with the amino acid sequences of glycine oxidases derivedfrom other thermophilic bacteria belonging to the family Bacillus, asdescribed above.

Therefore, as will be explained in the examples described later, when anattempt was made to introduce a mutation into this characteristicregion, it has been revealed that, in addition to the fact that, in themutant glycine oxidase in which the 251st glycine was substituted withanother amino acid, the enzyme activity is improved more than in thewild-type, the thermal stability is improved rather than beingmaintained, and that the kinetic constant (Michaelis constant) K_(m) isalso lower than that of the wild-type.

Accordingly, it can be said that the mutant glycine oxidase according tothe present disclosure introduces a mutation into a characteristicregion that is widely conserved in the thermophilic bacteria belongingto the family Bacillus, with respect to the wild-type glycine oxidasederived from the thermophilic bacteria belonging to the family Bacillus,whereby not only the enzyme activity is improved, but also unexpectedand excellent enzyme properties are obtained.

Therefore, the thermophilic bacteria belonging to the family Bacillusfrom which the mutant glycine oxidase according to the presentdisclosure is derived may be any bacteria as long as they are classifiedas Bacillaceae and have thermophilicity, as described above. Typicalexamples thereof include thermophilic bacteria belonging to the genusGeobacillus, and a more preferred example includes Geobacilluskaustophilus.

Further, as described above, the thermophilic bacteria belonging to thegenus Geobacillus include, in addition to G. kaustophilus, G.stearothermophilus, G. thermoleovorans, G. thermodenitrificans and G.subterraneus, Geobacillus thermoglucosidasius, Geobacilluscalboxylosilyticus, Geobacillus tepidamans, Geobacillus galactosidasius,Geobacillus zalihae, other unclassified strains of the genus Geobacillus(Geobacillus sp.), and the like, but are not particularly limited.

Here, for example, in Reference Literature 2: Messele Yohannes EQUAR,Yasushi TANI, Hisaaki MIHARA “Purification and Properties of GlycineOxidase from Pseudomonas putida KT2440” Journal of Nutritional Scienceand Vitaminology, Vol. 61 pp. 506-510 (2015), a phylogenetic analysis ofglycine oxidase homology has been shown, and based on this analysis, ithas been revealed that the genera Bacillus, Alicyclobacillus,Brevibacillus, Geobacillus, Sulfobacillus, Paenibacillus, andSalinicoccus had differentiated from the same strain. In other words, itcan be seen that there is a high possibility that the characteristicregion is preserved, at least in glycine oxidases derived from each ofthe genus described above, among the Bacillus bacteria.

Therefore, in the present disclosure, a more preferable example of thethermophilic bacteria belonging to the family Bacillus from whichglycine oxidase is derived can include, other than the genusGeobacillus, thermophilic bacteria belonging to the genus Bacillus, thegenus Alicyclobacillus, the genus Brevibacillus, the genusSulfobacillus, the genus Paenibacillus, or the genus Salinicoccus.

[Mutant Glycine Oxidase]

The mutant glycine oxidase according to the present disclosure isobtained by introducing a mutation into a wild-type glycine oxidasederived from thermophilic bacteria belonging to the family Bacillus, asdescribed above, and it may be any one having the characteristic enzymeproperties shown in (1) to (7) below, as also shown in the examplesdescribed later.

(1) The molecular weight is 40,000±2,000 daltons in SDS-polyacrylamidegel electrophoresis (SDS-PAGE).

(2) The optimum temperature is 45° C. under the condition of pH 8.5 inpresence of pyrophosphate.

(3) The optimum pH is pH 8.0 under the condition of 37° C. in presenceof pyrophosphate.

(4) Thermal stability is stable up to 70° C. under the condition of pH8.5 while retaining for 1 hour in presence of pyrophosphate.

(5) pH Stability is stable in the range of pH 5.5 to 10.0 under thecondition of 4° C. while retaining for 24 hours in presence ofpyrophosphate.

(6) The specific activity is 1.2 units/mg or more.

(7) The kinetic constant (Michaelis constant) K_(m) is 0.2 mM or less.

Here, among the above enzyme properties, (1) the molecular weight isbased on the molecular weight of wild-type glycine oxidase, but may bewithin the range of 39,000±1,000 daltons. In addition, (6) the specificactivity is preferably 2.4 units/mg or more, and more preferably 4.0units/mg or more.

In the present disclosure, the enzyme properties (1) to (7) can beevaluated based on “Evaluation of Various Properties of Glycine Oxidase”in the examples described later, and may be evaluated by other knownmethods.

More specific mutant glycine oxidase can include the mutant glycineoxidase derived from the Geobacillus kaustophilus HTA426 straindescribed in detail in the examples, as described above. This mutantglycine oxidase can include those having an amino acid sequence in whicha 251st amino acid in the amino acid sequence represented by SEQ ID NO:1 is substituted from glycine to another amino acid, as shown in FIG. 1, that is, those having an amino acid sequence shown in SEQ ID NO: 3 forsequence. In SEQ ID NO: 3, the 251st (position 251) amino acid isindicated by an arbitrary amino acid Xaa, and FIG. 1 shows that it issubstituted with an arbitrary amino acid X by an arrow.

Of course, the mutant glycine oxidase according to the presentdisclosure is not limited to one in which the 251st (position 251)glycine in the amino acid sequence shown in SEQ ID NO: 1 is mutated(having the amino acid sequence of SEQ ID NO: 3), and it may be any onehaving the characteristic enzyme properties of (1) to (7) above.However, when SEQ ID NO: 1 is used as a reference, it may be any onehaving 95% or more homology (or a sequence identity), more preferablyany one having 97% or more homology, and further preferably any onehaving 98% or more homology to the amino acid sequence shown in SEQ IDNO: 1.

In addition, the mutant glycine oxidase according to the presentdisclosure, as explained in the examples described later, when SEQ IDNO: 1 is used as a reference, it is preferable that the 251st (position251) glycine is one having an amino acid sequence substituted withanother basic amino acid or a hydrophobic amino acid. Thereby, it ispossible to achieve better enzyme activity as compared with thewild-type glycine oxidase. Specific basic amino acids are notparticularly limited, and can include glutamine, arginine, histidine, orthe like. Also, specific hydrophobic amino acids are not particularlylimited, and can include isoleucine or threonine. Alternatively, asexplained in the examples described later, another amino acid may be anyone of alanine, glutamic acid, histidine, isoleucine, asparagine,glutamine, arginine, or threonine.

As described above, the mutant glycine oxidase according to the presentdisclosure is one that achieves the above enzyme properties byintroducing a mutation into the wild-type glycine oxidase derived fromthermophilic bacteria belonging to the family Bacillus. This mutantglycine oxidase can maintain a kinetic constant K_(m) that issubstantially in the same range as that of the conventional mutantglycine oxidase, also, high thermal stability can be achieved ascompared not only with the conventional mutant glycine oxidase but alsowith the wild-type glycine oxidase, and furthermore, high specificactivity can be achieved as compared with the conventional mutantglycine oxidase and the wild-type glycine oxidase. Therefore, accordingto the present disclosure, it is possible to obtain a mutant glycineoxidase capable of achieving good thermal stability as well as goodenzyme activity.

[DNA Encoding Mutant Glycine Oxidase]

The present disclosure also includes DNA encoding the mutant glycineoxidase of the above configuration. SEQ ID NO: 2 in the sequence listingshows a base sequence encoding glycine oxidase derived from theGeobacillus kaustophilus HTA426 strain, and a typical example of the DNAaccording to the present disclosure include a DNA having a base sequenceinto which a mutation has been introduced in the base sequence shown inSEQ ID NO: 2.

As such a DNA, for example, as shown in FIG. 2 , a codon composed ofnucleotides 751 to 753 in the base sequence represented by SEQ ID NO: 2in the sequence listing is a DNA having a base sequence substituted froma codon of guanine-guanine-cytosine (GGC) encoding glycine to a codonencoding another amino acid, that is, a DNA having a base sequence shownin SEQ ID NO: 4 in the sequence listing. In SEQ ID NO: 4, thenucleotides 751 to 753 encoding the 251st amino acid are indicated by nas an arbitrary nucleotide, and FIG. 2 shows they are substituted withan arbitrary nucleotide N by an arrow.

As described above, a DNA having the base sequence shown in SEQ ID NO: 2is a DNA encoding the wild-type glycine oxidase derived from theGeobacillus kaustophilus HTA426 strain. Accordingly, the DNA having thebase sequence shown in SEQ ID NO: 4 can be said to be a DNA in which thecodon corresponding to the 251st glycine is substituted with a codonencoding another amino acid in the DNA encoding the wild-type glycineoxidase.

Here, the DNA according to the present disclosure is not limited to theDNA having the base sequence shown in SEQ ID NO: 4, and for example, maybe a DNA having a base sequence homologous to the base sequence shown inSEQ ID NO: 4, or a DNA having another base sequence encoding the aminoacid sequence shown in SEQ ID NO: 3. Further, the DNA according to thepresent disclosure may be a mutant DNA in which a mutation is introducedat a position excluding a codon corresponding to the 251st amino acid inthe base sequence shown in SEQ ID NO: 4. This mutant DNA includes thosehaving a base sequence in which one or two or more bases are deleted,substituted or added in the base sequence shown in SEQ ID NO: 4 withinthe range that retains an activity of a mutant glycine oxidase to beencoded. The number of bases to be deleted, substituted or added isusually within the range of 1 to 120, preferably within the range of 1to 60, and more preferably within the range of 1 to 30.

The present disclosure also includes a replicable recombinant DNAcontaining a DNA encoding the mutant glycine oxidase of the aboveconfiguration and an autonomously replicable vector. A typical exampleof such an autonomously replicable vector includes a plasmid vector.

Specific plasmid vectors include pBR plasmids such as pBR322; pUCplasmids such as pUC18, pUC19, pUC118, and pUC119; pBS plasmids such aspBlueScript II, pBluescript II SK(+/−), pBluescript II KS(+/−),pBluescript II XR, and pBluescript II RI; pET plasmids such as pET-3a to3d, pET-11a to d, pET-14b, pET-15b, and pET-21a to 21d; pGEX plasmidssuch as pGEX-1, pGEX-2T, and pGEX-3X; pTZ plasmids such as pTZ4, pTZ5,pTZ12, pTZ-18R, and pTZ-19R; pSU plasmids such as pSUO, pSU7, pSU22, andpSU23; genus Bacillus plasmids such as pUB110, pC194, pHY plasmids, pNUplasmids, pNY326, and pNC plasmids; shuttle vector plasmids such aspHV14, TRp7, YEp plasmids, and pBS7; and the like.

These plasmids can be appropriately selected according to variousconditions such as the type of cell serving as a host and the type ofexpression system. In addition, the autonomously replicable vector maybe a phage vector or the like.

The method for inserting the DNA encoding the mutant glycine oxidase ofthe above configuration into an autonomously replicable vector is notparticularly limited, and a known method can be suitably used. Generalexamples include a method of digesting (cutting) a DNA (or gene)encoding a mutant glycine oxidase and a vector with a known type IIrestriction enzyme, and annealing these DNA fragments and vectorfragments as necessary, then ligating using DNA ligase and the like, butare not particularly limited.

The replicable recombinant DNA of the above configuration may containthe DNA encoding the mutant glycine oxidase of the above configurationand a DNA other than the autonomously replicable vector. For example, aDNA encoding a control sequence not contained in the autonomouslyreplicable vector may be contained, or a DNA (or gene or the like)encoding another protein or peptide may be contained. At this time, themutant glycine oxidase of the above configuration may be incorporatedinto a replicable recombinant DNA so as to constitute a chimeric proteintogether with other proteins and peptides.

[Method for Producing Mutant Glycine Oxidase, Etc.]

Such recombinant DNA can be introduced into cells that serve as knownhosts. Accordingly, the present disclosure also includes a transformantobtained by introducing a replicable recombinant DNA containing a DNAencoding the mutant glycine oxidase of the above configuration and anautonomously replicable vector into a host cell. Examples of the hostcells generally include microorganisms such as Escherichia coli,Bacillus subtilis, actinomycetes, and yeast, but are not limitedthereto, and may be plant cells or animal cells.

In the examples described later, Escherichia coli is used for both thehost cell for replicating the replicable recombinant DNA and the hostcell for producing the mutant glycine oxidase of the above configurationfrom the replicated recombinant DNA. However, the present disclosure isnot limited thereto, and for example, when replicating a replicablerecombinant DNA, Escherichia coli may be used as the host cell, and whenproducing the mutant glycine oxidase of the above configuration,Bacillus subtilis, that is, bacteria belonging to the genus Bacillus maybe used as the host cell. Also, in recent years, a protein expressionsystem using bacteria belonging to the genus Brevibacillus, which is oneof Bacillus bacteria, has been constructed and marketed. Therefore, whenproducing the mutant glycine oxidase of the above configuration,bacteria belonging to the genus Brevibacillus may be used as the hostcell.

The method for introducing the recombinant DNA of the aboveconfiguration into the host cell, that is, the transformation method, isnot particularly limited, and a known method according to the type ofthe host cell or the type of the autonomously replicable vector can beused. In the case of bacteria such as Escherichia coli, examples oftypical transformation methods can include an electroporation method, amethod of making cells into competent cells using calcium chloride, andthe like. In the case where the host cell is yeast, a method ofpartially removing cell walls of the yeast cells to make spheroplast, alithium acetate method, or the like can be used. In addition, in thecase where the host cell is a fungus, a plant cell, or an animal cell, aparticle gun method, a transfection method or the like can also be used.

Furthermore, the present disclosure includes not only a transformantobtained by introducing a recombinant DNA containing the DNA encodingthe mutant glycine oxidase of the above configuration and anautonomously replicable vector into the host cell, but also cells inwhich the DNA encoding the mutant glycine oxidase of the aboveconfiguration has been introduced into a genome of the host cell. Themethod for introducing the DNA encoding the mutant glycine oxidase ofthe above configuration into the genome of the host cell is notparticularly limited, and for example, when the host cell isSaccharomyces cerevisiae or its related species, the DNA can beintegrated into a chromosome, using a YIp plasmid. Also, regardless ofthe type of the host cell, the DNA can be integrated into a chromosomeor the like using a genome editing technique.

The transformant or DNA-integrated cell thus obtained may be culturedusing a known nutrient medium according to various conditions such asthe type of the host cell or the purpose of culture. For example, whenreplicating recombinant DNA using Escherichia coli as a host, an LBmedium (LB broth) or the like may be used. Moreover, in the case ofproducing the mutant glycine oxidase of the above configuration byculturing the transformant of the above configuration or theDNA-integrated cell of the above configuration, a known culture medium(broth) corresponding to the type of the transformant or cell may beused. Further, various known additional components may be added to aculture medium according to various conditions.

As described above, the mutant glycine oxidase according to the presentdisclosure can be produced by introducing (or integrating) the DNAencoding the mutant glycine oxidase into a host cell by various methodsto prepare a transformant (or DNA-integrated cell), and culturing theobtained transformant (or DNA-integrated cell). Therefore, the presentdisclosure also includes a method for producing a mutant glycineoxidase, including culturing such cells and collecting the mutantglycine oxidase of the above configuration from the obtained culture.

In the method for producing a mutant glycine oxidase according to thepresent disclosure, a cell culture scale is not particularly limited.For example, when a liquid medium (broth) is used, it may be asmall-scale culture using a test tube or a flask, may be a large-scaleculture using a jar fermenter, or may be a large-scale culture using atank at an industrial level.

A method for collecting the mutant glycine oxidase of the aboveconfiguration from the cultured cells is not particularly limited, and aknown method can be used. When the expressed mutant glycine oxidaseaccumulates in the cells, the mutant glycine oxidase may be collected bycollecting the cultured cells, disrupting the cells by a known method toobtain a crude enzyme solution, and purifying or concentrating the crudeenzyme solution by a known method. When it is not necessary to purify orconcentrate, the above crude enzyme solution may be used as the mutantglycine oxidase according to the present disclosure. In addition, whenthe expressed mutant glycine oxidase is significantly secretedextracellularly, the mutant glycine oxidase may be collected from theentire culture including the cultured cells and the broth.

Thus, the mutant glycine oxidase according to the present disclosure canmaintain a kinetic constant K_(m) that is substantially in the samerange as that of the conventional mutant glycine oxidase, also, highthermal stability can be achieved as compared not only with theconventional mutant glycine oxidase but also with the wild-type glycineoxidase, and further achieves high specific activity as compared withthe conventional mutant glycine oxidase and the wild-type glycineoxidase. Therefore, the mutant glycine oxidase according to the presentdisclosure can be suitably used for enzyme measurement of glycine.

Further, the mutant glycine oxidase according to the present disclosurecan be easily produced by introducing a DNA encoding the mutant glycineoxidase into a host cell, or the like, as described above. Therefore, itis also possible to mass-produce the mutant glycine oxidase according tothe present disclosure at an industrial level, and also possible tostably provide, for example, as a reagent for enzyme measurement ofglycine, a reagent for automatic measurement system of glycine, or thelike.

EXAMPLES

The present invention will be described more specifically based onexamples and comparative examples, but the present invention is notlimited thereto. Those skilled in the art can make various changes,modifications, and alterations without departing from the scope of thepresent invention. Here, Bacillus bacteria used in the followingexamples and comparative examples, a cloning method, enzyme activitymeasurement and the like were performed as shown below.

(Bacillus Bacteria)

As Bacillus bacteria derived from a wild-type glycine oxidase in thisexample, Geobacillus kaustophilus HTA426 strain was used (forconvenience of explanation, the Geobacillus kaustophilus HTA426 strainis hereinafter abbreviated as “HTA426”). HTA426 is available, forexample, from the National Research and Development Agency, RIKENBioResource Research Center (RIKEN BRC) Microbe Division (JCM)(JCM12893).

(Cloning of Glycine Oxidase)

Cloning of glycine oxidase from HTA426 was performed according to themethod described in Non-PTL 1. However, a plasmid vector of pET-15b wasused as a vector, and E. coli BL21 (DE3) was used as a host cell (host).

(Measurement of Enzyme Activity of Glycine Oxidase)

The enzyme activity of glycine oxidase was also measured according tothe method described in Non-PTL 1.

Here, an assay buffer composed of 1 mM sodium pyrophosphate (pH 8.5), 5mM 4-aminoantipyrine, and 20 mM phenol was used, and the optimalconditions were set to 37° C. in presence of 10 mM pyrophosphate and 1mM glycine (substrate).

The method for measuring the enzyme activity of glycine oxidase in thisexample, that is, the method for measuring enzyme activity performedaccording to the method described in Non-PTL 1 using the above assaybuffer is referred to as “this glycine oxidase activity measurementmethod”.

(Evaluation of Various Properties of Glycine Oxidase)

Various properties of glycine oxidase were evaluated according to theconditions and/or methods described below.

(1) The molecular weight was determined by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) according to the method described in Non-PTL1.

(2) The optimum temperature was evaluated by varying measurementtemperature within the range of 25° C. to 45° C. in this glycine oxidaseactivity measurement method.

(3) The optimum pH was evaluated by varying pH using a 10 mM phosphatebuffer (pH 7.0 to 7.5), a 10 mM HEPES buffer (pH 7.5 to 8.0), a 10 mMpyrophosphate buffer (pH 8.0 to 8.5), and a pyrophosphate and carbonatemixed buffer (5 mM pyrophosphate and 5 mM carbonate buffer, pH 9.0 to10.0), respectively, in place of the assay buffer, in this glycineoxidase activity measurement method.

(4) Thermal stability was determined by varying reaction temperaturewithin the range of 30° C. to 90° C., and incubating for 15 minutes or 1hour, then measuring the remaining enzyme activity, in this glycineoxidase activity measurement method, and evaluated as a relativeactivity based on the enzyme activity at 37° C.

(5) pH Stability was determined by using a 100 mM phosphate buffer (pH5.5 to 7.5), a 10 mM pyrophosphate buffer (pH 8.0 to 8.5), and apyrophosphate and carbonate mixed buffer (5 mM pyrophosphate and 5 mMcarbonate buffer, pH 9.0 to 10.0), in place of the assay buffer, andincubating under the condition of 4° C. for 24 hours, then measuring theremaining enzyme, in this glycine oxidase activity measurement method,and evaluated as a relative activity based on the enzyme activity at pH8.5.

(6) The specific activity was calculated by dividing the enzyme activity(unit: unit [U]) of the enzyme sample used in this glycine oxidaseactivity measurement method by the enzyme mass (unit: mg) contained inthe enzyme sample. The enzyme mass is the mass of a protein contained inthe enzyme sample, and was quantified by measuring the absorbance at 595nm by Bradford method.

(7) The kinetic constant (Michaelis constant) K_(m) was calculated basedon Michaelis-Menten equation, by varying substrate concentration andmeasuring the enzyme activity, in this glycine oxidase activitymeasurement method.

Comparative Example 1

The specific activity, thermal stability, and kinetic constant K_(m) ofthe wild-type glycine oxidase derived from HTA426 were evaluated asdescribed above. The results are shown in Table 1, the specific activityis shown in FIG. 3A, and thermal stability is shown in FIG. 3B.

The amino acid sequence of the wild-type glycine oxidase is shown in SEQID NO: 1 in FIG. 1 and the sequence listing, and the base sequence ofthe gene (DNA) encoding the wild-type glycine oxidase is shown in FIG. 2and SEQ ID NO: 2 in the sequence listing. For convenience ofexplanation, the wild-type glycine oxidase is abbreviated as “WT-GOX”.

Comparative Example 2

The specific activity, thermal stability, and kinetic constant K_(m) ofa commercially available glycine oxidase, product number H244Kmanufactured by BioVision, Inc, were evaluated as described above. Theresults are shown in Table 1, the specific activity is shown in FIG. 3A,and thermal stability is shown in FIG. 3B.

Here, H244K is a mutant enzyme in which a mutation is introduced into awild-type glycine oxidase derived from B. subtilis, a Bacillus bacteriumthat is not a thermophilic bacterium, and the introduction of mutationis performed by the method described in Non-PTL 2.

TABLE 1 Specific Thermal Kinetic Enzyme activity stability constantK_(m) Comparative WT-GOX 0.7 U/mg Up to 60° C. 0.25 mM Example 1Comparative H244K 1.2 U/mg Up to 45° C. 0.14 mM Example 2 Example 1V-GOX1 4.0 U/mg Up to 70° C. 0.20 mM

Example 1

As described in the above embodiment, in WT-GOX of Comparative Example1, it was revealed that a 248th (position 248) to 255th (position 255)partial amino acid sequence in the amino acid sequence represented bySEQ ID NO: 1 is a characteristic region unique to thermophilic bacteriabelonging to the family Bacillus. Therefore, a mutation was introducedinto the characteristic region of WT-GOX based on the method describedin Non-PTL 2, and a plasmid vector containing a glycine oxidase geneinto which a mutation was randomly introduced was obtained. Forconvenience of explanation, the mutant glycine oxidase is abbreviated as“V-GOX” in this example.

Thus, the plasmid vector was introduced into the above host cell (E.coli BL21 (DE3)) based on the method described in Non-PTL 1, andexpression of V-GOX was confirmed. When the enzyme activity wasconfirmed for 204 types among the obtained colonies, 14 types ofcolonies expressing V-GOXs showing high activity were confirmed.Therefore, these 14 types of V-GOXs were sequentially analyzed, andtheir enzyme activities were measured.

As a result, as shown in Table 2, 8 types of V-GOXs (V-GOX1 to V-GOX8)could be identified. In these V-GOXs, the 251st (position 251) aminoacid in the amino acid sequence represented by SEQ ID NO: 1 issubstituted from glycine (Gly, G) to another amino acid. The enzymeactivity in Table 2 is shown as the ratio (times) of the enzyme reactionrate (use of crude enzyme solution) of V-GOX when WT-GOX is used asreference (1).

TABLE 2 Enzyme Codon Amino acid Enzyme activity (times) V-GOX1 CAAGlutamine (Gln, Q) 8.2 V-GOX2 ATA Isoleucine (Ile, I) 7.3 V-GOX3 CGTArginine (Arg, R) 5.1 V-GOX4 CAC Histidine (His, H) 4.8 V-GOX5 ACAThreonine (Thr, T) 4.5 V-GOX6 GCC Alanine (Ala, A) 3.4 V-GOX7 AACAsparagine (Asn, N) 3.3 V-GOX8 GAG Glutamic acid (Glu, E) 2.6 WT-GOX GGCGlycine (Gly, G) 1

Among these V-GOX1 to V-GOX8, V-GOX1 having an amino acid sequence inwhich the 251st (position 251) amino acid was substituted from glycine(Gly, G) to glutamine (Gln, Q) showed the highest enzyme activity.Therefore, after purifying this V-GOX1, the molecular weight (SDS-PAGE),the optimum temperature, the optimum pH, thermal stability, pHstability, the specific activity, and the kinetic constant K_(m) wereevaluated. The results of the specific activity, thermal stability, andthe kinetic constant K_(m) are shown in Table 1 above, the specificactivity is shown in FIG. 3A, and thermal stability is shown in FIG. 3B,and further, evaluation results of enzyme properties of V-GOX1containing them are also shown in Table 3 together with the conditions.

TABLE 3 Enzyme properties Conditions Numerical value Molecular weightSDS-PAGE 40 kDa Optimum temperature PPi, pH 8.5, reacted for 15 minutes45° C. Optimum pH PPi, 40° C., reacted for 15 minutes pH 8.0 Thermalstability PPi, pH 8.5, retained for 1 hour Up to 70° C. pH StabilityPPi, 4° C., reacted for 24 hours pH 5.5-10.0 Specific activity — 4.0U/mg Kinetic constant K_(m) — 0.2 mM * PPi: Pyrophosphate

(Comparison Between Examples and Comparative Examples)

As is clear from Table 1 and FIGS. 3A and 3B, the enzyme of ComparativeExample 1, that is, WT-GOX derived from HTA426 has thermal stability 15°C. higher as compared with the enzyme of Comparative Example 2, that is,H244K, a commercially available mutant glycine oxidase derived fromBacillus subtilis that is not a thermophilic bacterium, and althoughWT-GOX exhibits excellent thermal stability, its specific activity isgreatly inferior. Further, regarding the kinetic constant K_(m), theWT-GOX of Comparative Example 1 is higher than H244K of ComparativeExample 2.

In contrast, V-GOX1, which had the highest activity among the enzymes ofExample 1, had a specific activity 5.7 times that of the WT-GOX ofComparative Example 1 and 3.3 times that of the H244K of ComparativeExample 2. Further, thermal stability of V-GOX1 is 10° C. higher thanthat of the WT-GOX of Comparative Example 1, and 25° C. higher than thatof the H244K of Comparative Example 2. Moreover, the kinetic constantK_(m) of V-GOX1 is a value lower than that of the WT-GOX of ComparativeExample 1, and it can be said that it is a value comparable to the valueof the H244K of Comparative Example 2.

In addition, as is clear from Table 2, among WT-GOX amino acidsequences, the mutant enzymes V-GOX1 to V-GOX8 obtained by introducing amutation into the characteristic region of thermophilic bacteriabelonging to the family Bacillus all have higher enzyme activity thanWT-GOX. Even V-GOX8, which has the lowest enzyme activity among theseeight types, has a reaction rate 2.5 times or more that of WT-GOX.

Also, V-GOXs2 to 8 have substantially the same enzyme properties asV-GOX1 shown in Table 3, although not specifically shown. Therefore, notonly V-GOX1, but also seven types of enzymes, V-GOXs2 to 8, have bothexcellent enzyme activity and excellent thermal stability, and also havea value of kinetic constant K_(m) that does not greatly differ fromthose of conventional mutant enzymes. Therefore, it can be seen thatV-GOXs1 to 8 are characteristic mutant enzymes having enzyme propertiesas shown in Table 3 as compared with WT-GOX.

Furthermore, as is clear from Table 2, in V-GOXs1 to 8, glycine (G), the251st (position 251) amino acid of WT-GOX is substituted with any ofvarious amino acids, that is alanine (A), glutamic acid (E), histidine(H), isoleucine (I), asparagine (N), glutamine (Q), arginine (R), orthreonine (T). Therefore, it can be seen that V-GOX having higheractivity than WT-GOX is obtained by substituting glycine with an aminoacid other than glycine. In particular, it can be also seen that highlyactive V-GOX can be easily obtained by substituting glycine with any ofthe above eight types.

In addition, amino acids substituted from glycine in V-GOXs1 to 5 havingrelatively high enzyme activity are basic or hydrophobic amino acids.That is, glutamine, which is a substituted amino acid of V-GOX1,arginine, which is a substituted amino acid of V-GOX3, and histidine,which is a substituted amino acid of V-GOX4, are all basic amino acids,and isoleucine, which is a substituted amino acid of V-GOX2, andthreonine, which is a substituted amino acid of V-GOX5, are bothhydrophobic amino acids. Therefore, it can be seen that the amino acidsubstituted from glycine in WT-GOX is preferably a basic amino acid or ahydrophobic amino acid.

It should be noted that the present invention is not limited to thedescription of the above-described embodiment, and various modificationsare possible within the scope shown in the scope of the claims, and aredisclosed in different embodiments and a plurality of modifications.Embodiments obtained by appropriately combining the technical means arealso included in the technical scope of the present invention.

In addition, from the above description, many modifications and otherembodiments of the present invention are obvious to those skilled in theart. Accordingly, the foregoing description should be construed asillustrative only and is provided for the purpose of teaching thoseskilled in the art the best mode of carrying out the invention. Thedetails of the structure and/or function can be modified substantiallywithout departing from the spirit of the invention.

INDUSTRIAL APPLICABILITY

The present invention can be widely used suitably in the field relatedto the mutant glycine oxidase that achieves both good enzyme activityand good thermal stability, obtained by introducing a mutation into aglycine oxidase derived from thermophilic bacteria of the familyBacillus, including thermophilic bacteria of the genus Bacillus or thegenus Geobacillus.

The invention claimed is:
 1. A mutant glycine oxidase which is a mutantenzyme in which a glycine at the 251st position of an amino acidsequence having at least 95% sequence identity to SEQ ID NO: 1, in anamino acid motif of asparagine (N), glycine (G), cysteine (C), andtyrosine (Y), of a wild-type glycine oxidase derived from thermophilicbacteria selected from a group consisting of Geobacillus kaustophilus,Geobacillus stearothermophilus, and Geobacillus thermoleovorans issubstituted with another amino acid, the mutant glycine oxidase having:a molecular weight of 40,000±2,000 daltons in sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE); an optimumtemperature of 45° C. under a condition of pH 8.5 in presence ofpyrophosphate; an optimum pH of pH 8.0 under a condition of 37° C. inpresence of pyrophosphate; a thermal stability being stable up to 70° C.under a condition of pH 8.5 while retaining for 1 hour in presence ofpyrophosphate; a pH stability being stable in a range of pH 5.5 to 10.0under a condition of 4° C. while retaining for 24 hours in presence ofpyrophosphate; a specific activity of at least 1.2 units/mg; and akinetic constant (Michaelis constant) K_(m) of 0.2 mM or less.
 2. Amutant glycine oxidase which is a mutant enzyme of glycine oxidasederived from thermophilic bacteria belonging to the family Bacillus, themutant glycine oxidase having at least 95% sequence identity to SEQ IDNO: 1, and the mutant glycine oxidase having an amino acid sequence inwhich a 251st amino acid in SEQ ID NO: 1 is substituted from glycine toanother amino acid.
 3. The mutant glycine oxidase according to claim 2,in which the 251st amino acid is substituted from glycine to a basicamino acid or a hydrophobic amino acid.
 4. The mutant glycine oxidaseaccording to claim 3, wherein the basic amino acid is glutamine,arginine, or histidine, and the hydrophobic amino acid is isoleucine orthreonine.
 5. The mutant glycine oxidase according to claim 2, whereinthe another amino acid is alanine, glutamic acid, histidine, isoleucine,asparagine, glutamine, arginine, or threonine.
 6. A DNA sequenceencoding the mutant glycine oxidase according to claim
 1. 7. Areplicable recombinant vector comprising the DNA sequence according toclaim 6 and an autonomously replicating sequence.
 8. A cell expressingthe mutant glycine oxidase obtained by introducing the DNA sequenceaccording to claim 6 into a host cell.
 9. A method for producing amutant glycine oxidase, comprising culturing the host cell according toclaim 8, and collecting the mutant glycine oxidase from the resultingculture.
 10. A cell obtained by introducing the recombinant DNA vectoraccording to claim 7 into a host cell.
 11. The mutant glycine oxidaseaccording to claim 2, wherein The thermophilic bacteria belonging to thefamily Bacillus is a bacteria belonging to one of genus Bacillus, genusAlicyclobacillus, genus Brevibacillus, genus Geobacillus, genusSulfobacillus, genus Paenibacillus, and genus Salinicoccus.
 12. A DNAsequence encoding the mutant glycine oxidase according to claim
 2. 13. Areplicable recombinant vector comprising the DNA sequence according toclaim 12 and an autonomously replicating sequence.
 14. A cell obtainedby introducing the DNA sequence according to claim
 12. 15. A method forproducing a mutant glycine oxidase, comprising culturing the cellaccording to claim 14, and collecting the mutant glycine oxidase fromthe resulting culture.
 16. A cell obtained by introducing therecombinant DNA vector according to claim 13 into a host cell.